实验性肺纤维化大鼠肺组织整合素α5β1和转化生长因子-β的表达

目的　研究纤维连接蛋白受体整合素α５β１ 在大鼠肺纤维化中的作用。方法　用免疫组化方法观察实验性大鼠肺纤维化纤维连接蛋白（ＦＮ） 及其受体整合素α５β１ 和转化生长因子β（ＴＧＦβ）表达的动态变化;用Ｎｏｒｔｈｅｒｎ 印迹杂交和免疫细胞化学方法观察ＴＧＦβ对体外培养的大鼠肺成纤维细胞整合素α５β１ ｍＲＮＡ和蛋白表达的影响。结果　（１）实验组１～３ 天,病灶内上皮细胞和内皮细胞整合素α５β１ 表达明显增强,炎细胞及增生的间质细胞亦呈阳性反应。以后,整合素α５β１ 阳性反应主要见于明显增生的间质细胞,并与ＴＧＦβ阳性分布相似。ＦＮ的变化与整合素α５β１ 的变化同步。（２）ＴＧＦβ作用后,大鼠肺成纤维细胞整合素α５β１ ｍＲＮＡ 及其蛋白表达增强（α５、β１ ｍＲＮＡ 杂交条带的积分光密度值,依次分别是对照的６．６ 、４．４ 倍;α５β１ 蛋白染色积分光密度值为对照的３．６ 倍） 。结论　整合素α５β１在肺间质细胞活化、增殖、分化及细胞外基质合成中起了极其重要作用。     

大鼠肝纤维化Ito细胞纤维连接蛋白受体α5β1的表达

目的研究纤维连接蛋白受体α５β１在大鼠肝纤维化中的作用。方法应用Ｎｏｒｔｈｅｒｎ印迹杂交及免疫组化方法观察大鼠实验性纤维化肝组织和离体Ｉｔｏ细胞纤维连接蛋白（ＦＮ）及其受体α５β１表达的变化。结果（１）α５β１主要分布于血窦壁的内皮细胞和部分结蛋白（ＤＭ）阳性细胞（Ｉｔｏ细胞）。实验组ＤＭ阳性细胞α５β１表达增强，１０周时达高峰。ＦＮ的变化与之同步。（２）实验组肝组织ＦＮ、α５和β１ｍＲＮＡ含量增高，峰值在第６周；离体Ｉｔｏ细胞三种ｍＲＮＡ含量实验组高于对照组。结论结果提示：Ｉｔｏ细胞表达α５β１，肝纤维化时Ｉｔｏ细胞的激活，可导致ＦＮ、α５和β１ｍＲＮＡ表达增强，应用Ｎｏｒｔｈ－ｅｒｎ印迹杂交技术检测ＦＮ及其受体ｍＲＮＡ的表达，从基因水平了解Ｉｔｏ细胞激活和增生状态，能更敏感地反映肝纤维化的发展倾向。     

大鼠肝纤维化Ito细胞纤维连接蛋白受体α5β1的表达

研究纤维连接蛋白受体α５β１在大鼠肝纤维化中的作用。     

肿瘤坏死因子对晶状体上皮细胞的粘附移行作用

目的：探讨肿瘤坏死因子α（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ α,ＴＮＦ－０６３３）对晶状体上皮细胞在细胞外基质上的粘附移行作用。以及在此过程中整合素分子的表达情况。方法：借助细胞粘附实验和移行实验研究ＴＮＦ－α对体外培养的牛晶状体上皮细胞在细胞外基质上的粘附和移行,以及整合素抗体封闭后对细胞的影响。ＴＮＦ－α对细胞表面整合素的表达调节经免疫荧光染色后由流式细胞仪检测。结果：ＴＮＦ－α可以明显促进晶状体上皮细胞在Ⅳ型胶原、层粘蛋白、纤维连接蛋白上的粘附移行。抗体封闭实验表明整合素β１、α２、α５亚基参与细胞的粘附,在细胞移行中发挥主要作用的是α亚基而非β亚基,流式细胞仪检测表明ＴＮＦ－α可上调晶状体上皮细胞中整合素的表达。结论：胶原、层粘蛋白、纤维连接蛋白是晶状体囊的主要成分。肿瘤坏死因子α促进晶状体上     

侧支血管整合素αvβ3的表达及细胞外基质对其表达的调节

目的:研究侧支血管生成过程中整合素αvβ3的表达及细胞外基质对其表达的调节。方法:结扎大鼠股动脉,应用免疫荧光组织化学技术检测侧支血管整合素αvβ3的表达;并在体外实验中检测不同细胞外基质成分对整合素αVβ3表达的影响。结果:侧支血管整合素αvβ3表达明显增强,为正常血管的3.47倍;种植在纤维连接蛋白(fibronectin,FN)上的血管平滑肌细胞(smooth muscle cells,SMC)整合素αvβ3的表达最高,层粘连蛋白(laminin,LN)上次之,三维Matrigel基膜成分胶里最低,差异具有显著性(P<0.001)。结论:相对于正常血管,侧支血管生整合素αvβ3的表达显著上调;不同细胞外基质成分对血管平滑肌细胞中整合素αvβ3表达的影响不同。     

人肝细胞癌中整合素α5,β1亚基和纤维连接蛋白mRNAs的表达

目的研究肝细胞癌（ＨＣＣ）中整合素α５、β１亚基和纤维连接蛋白（ＦＮ）ｍＲＮＡｓ表达的生物学意义。方法应用Ｎｏｒｔｈｅｒｎ印迹杂交和核酸原位杂交技术，对１５例ＨＣＣ癌组织、５例癌旁肝硬化和３例正常肝组织中整合素α５、β１亚基和ＦＮｍＲＮＡｓ表达作杂交分析。结果高分化ＨＣＣ癌组织中三种ｍＲＮＡｓ表达水平与癌旁及正常肝组织相近，而低、中分化ＨＣＣ癌组织内明显减少，甚至消失（分别Ｐ＜００１）；整合素α５亚基和ＦＮｍＲＮＡｓ主要见于癌细胞胞浆内；ＨＣＣ伴肝内浸润和／或转移组癌组织内三种ｍＲＮＡｓ水平较无浸润转移组显著下降（Ｐ＜００５）；５例伴肝内转移的ＨＣＣ癌组织中ＦＮｍＲＮＡ呈异质性表达。结论ＨＣＣ中整合素α５、β１和ＦＮ蛋白的表达变化系癌细胞基因转录水平下调的结果，可能与ＨＣＣ细胞分化、浸润和转移相关；异质性ＦＮｍＲＮＡ及其编码的ＦＮ蛋白可能在ＨＣＣ转移中有意义。     

大鼠后肢侧支动脉生成过程中整合素α5β1和纤维连接蛋白的表达

目的:探讨侧支动脉生成过程中平滑肌细胞(smooth muscle cells,SMC)移动增殖的机制。方法:结扎大鼠股动脉,应用免疫荧光组织化学技术检测侧支血管中整合素α5β1、纤维连接蛋白(fibronectin,FN)和层粘连蛋白(laminin,LN)的表达。并在体外实验中检测了不同的细胞外基质对SMC表达整合素α5β1的影响。结果:侧支血管整合素α5β1和FN的表达分别是对照侧的1.9倍和2倍;而LN的表达是对照侧的1／3。种植在FN上的SMC高表达整合素α5β1;种植在LN上的表达较低;而种植在三维Matrigel基膜成分胶里的SMC几乎不表达整合素α5β1。结论:在侧支血管发育过程中,整合素α5β1、FN和LN的表达变化,有助于侧支血管生长发育过程中SMC的迁移和增殖。     

甲氨喋呤对胶原诱导性关节炎大鼠滑膜骨桥蛋白和整合素αVβ3表达的影响

目的 探讨甲氨喋呤对胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠滑膜骨桥蛋白(osteopontin,OPN)及其受体整合素αvβ3表达的影响及其作用机制.方法 建立CIA大鼠模型,分为模型组和甲氨喋呤(MTX)组,后者用MTX进行干预治疗;采用免疫组织化学染色技术检测滑膜组织OPN和整合素αvβ3的表达,ELISA方法检测血清TNF-α水平.结果 ① CIA模型组和MTX组大鼠的滑膜OPN、整合素αvβ3表达均明显高于正常大鼠对照组(均为P<0.01);与模型组比较,MTX组OPN和整合素αVβ3的表达减少(均为P<0.01). ②正常对照组和MTX组大鼠血清TNF-α水平显著性低于模型组大鼠(均为P<0.01).结论 滑膜OPN和整合素αvβ3异常表达在CIA发病中具有重要意义.MTX通过下调滑膜OPN和整合素αvβ3的表达,降低血清TNF-α水平从而达到治疗CIA的作用.     

整合素α4β1及其配体与大鼠肝肿瘤周边肥大细胞募集的关系

目的：D整合素α4β1(VLA-4)主其配体VCAM－1（vascular cell adhesion molecular-1)和FN（fibronectin)与肝肿瘤周边肥大细胞（mast cell,MC)募集的关系。方法：根据肝肿瘤周边肥大细胞数量,将18只雄性Wistar大鼠移植肝肿瘤模型进行分析,8只正常雄性Wistar大鼠作对照,用间接免疫荧光和流式细胞术检测各级一大鼠腹腔肥大细胞整合素VLA－4分子的表达水平,同时用免疫组化研究肿瘤周边肝组织血管内皮细胞和肝窦内皮细胞表面VCAM－1和肿瘤周边FN的表达。结果：不同肝肿瘤大鼠肿瘤周边浸润肥大细胞数量有明显差异。各组大鼠腹腔MC表达整合素VLA－4分子均呈阳性,肿瘤周边肥大细胞浸润较多组,其整合素α4β1表达水平也较高。肿瘤周边血管内皮和窦内皮细胞表达VCAM－1阳性。肿瘤周边沉积大量呈阳性表达的FN与须大细胞紧密相联。结论：整合素α4β1及其配体VCAM－1和FN在肝肿瘤周边肥大细胞集要作用;整合素α4β1的表达水平与肿瘤周边MC数呈平行关系。     

血管紧张素Ⅱ对血管内皮细胞整合素β3表达的影响

目的和方法：本实验采用细胞ＥＬＩＳＡ和ＲＴ－ＰＣＲ的方法分别从蛋白质和ｍＲＮＡ水平对血管紧张素Ⅱ（Ａｎｇ－Ⅱ）作用下的内皮细胞表面整合素β３的表达进行检测。结果：１０－８ｍｏｌ／Ｌ～１０－５ｍｏｌ／Ｌ的Ａｎｇ－Ⅱ在蛋白质水平能够促进内皮细胞整合素β３表达（Ｐ＜０．０５），且呈剂量依赖性。１０－６ｍｏｌ／Ｌ的Ａｎｇ－Ⅱ作用培养的人脐静脉内皮细胞１８ｈ后，细胞整合素β３ｍＲＮＡ量为正常对照组的１５倍。结论：提示Ａｎｇ－Ⅱ促进内皮细胞表面整合素β３的表达可能是单核细胞和内皮细胞之间粘附功能增加的机制之一。     

肺纤维化大鼠中基质金属蛋白酶-2的表达

目的：研究基质金属蛋白酶－2（MMP2）在肺纤维化中的作用。方法：（1）用免疫组织化学方法（ABC法）观察博莱霉素所诱导的实验性大鼠纤维化肺组织内MMP2表达的动态变化;（2）用逆转录－聚合酶链反应（RT－PCR）观察博莱霉素所诱导的肺纤维化不同时期间质成纤维细胞MMP2 mRNA的动态变化。（3）用Northern印迹杂交和免疫细胞化学方法观察转化生长因子β1（TGFβ1）对体外培养的大鼠肺成纤维细胞MMP2mRNA和蛋白表达的影响。结果：（1）诱导纤维化组1－7d,病灶内浸润的单核巨噬细胞及肺泡间质细胞数量增多,MMP2免疫组织化学染色阳性,14d以后,阳性单核巨噬细胞减少,用博莱霉素28d时,阳性间质细胞亦减少。（2）博莱霉素作用后1d,肺成纤维细胞MMP2基因转录即明显增强,为对照组的2．05倍,28d时下降。（3）TGFβ1作用后,大鼠肺成纤维细胞MMP2mRNA及其蛋白表达增强。结论：肺组织内MMP2的过度表达,可能是肺纤维化启动的重要原因。     

Regulation of T-cell interaction with fibronectin by transforming growth factor-β is associated with altered Pyk2 phosphorylation

Although the involvement of transforming growth factor-beta (TGF-beta) in inflammatory reactions has been extensively studied, its mode of action in the context of the extracellular matrix (ECM) is still not fully understood. We undertook this study in an attempt to reveal the putative roles of TGF-beta in T-cell adhesion and migration. We found that a 60-min treatment of T cells with TGF-beta regulates T-cell adhesion to fibronectin (FN), a prototype cell adhesion protein of the ECM, depending on the presence of other activators. At 5 pg/ml to 1 ng/ml, TGF-beta alone induced T-cell adhesion to FN in an integrin alpha4/beta1- and integrin alpha5/beta1-dependent manner. TGF-beta also attenuated T-cell migration on the stromal cell-derived factor (SDF)-1alpha gradients. These effects of TGF-beta were not accompanied by alteration in the expression of very-late activation antigen type 4 (VLA-4) and VLA-5, nor were they mediated by the cyclo-oxygenase pathway. The cellular mechanism underlying the adhesion-regulating activities of TGF-beta involves adhesion-associated cytoskeletal elements. TGF-beta induced the phosphorylation of focal adhesion kinase Pyk2, but not extracellular signal-regulated kinase (ERK), and this effect was markedly increased in the presence of immobilized FN, suggesting a collaborative role for FN-specific integrins. Indeed, TGF-beta-induced Pyk2 phosphorylation was inhibited by monoclonal antibodies against VLA-4, VLA-5 and CD29. Thus, TGF-beta, which may appear at extravascular sites during inflammation, affects the adhesion of T cells to ECM glycoproteins and their migration by its ability to differentially induce or inhibit the phosphorylation of Pyk2.     

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.     

肝细胞癌整合蛋白α5β1,纤维连接蛋白及其降解片段的表达

目的探讨整合蛋白α５β１、纤维连结蛋白（ＦＮ）及其降解片段——恶性疾病相关性ＤＮＡ结合蛋白２（ＭＡＤ２）与肝细胞癌（ＨＣＣ）生物学行为间的关系及血浆ＭＡＤ２检测的意义。方法应用免疫组化及图像分析技术对４０例ＨＣＣ组织作整合蛋白α５β１、ＦＮ和ＭＡＤ２定位和定量分析,并以酶联免疫吸附法检测３０／４０例ＨＣＣ患者血浆中ＭＡＤ２和ＦＮ浓度。结果高分化ＨＣＣ组织整合蛋白α５β１、ＦＮ和ＭＡＤ２表达与正常及癌旁组织相近,而中、低分化ＨＣＣ中三者均明显减弱或消失;肿瘤浸润包膜处三种蛋白表达减少,但血管内癌栓外侧缘癌细胞整合蛋白α５β１和ＦＮ表达较强,并与相应血管内皮的强表达相映。ＨＣＣ患者血浆ＭＡＤ２平均浓度显著高于慢性肝病及正常对照者。结论整合蛋白α５β１和ＦＮ表达可能在ＨＣＣ分化、浸润及转移中起作用;ＨＣＣ患者血浆ＭＡＤ２含量的检测可能有一定临床意义。     

Expression of transforming growth factor-beta type I and type II receptors is altered in rat lungs undergoing bleomycin-induced pulmonary fibrosis

Transforming growth factor-beta (TGF-beta) is a family of autocrine/paracrine/endocrine cytokines involved in controlling cell growth and extracellular matrix metabolism. TGF-beta exerts its biological effects via binding to type I (TbetaRI) and type II (TbetaRII) receptors. To gain insight into the possible role of TGF-beta receptors in the pathogenesis of pulmonary fibrosis, we investigated the expression of TGF-beta receptors and their ligands in a bleomycin-induced model of pulmonary fibrosis. We found that the expression of both TbetaRI and TbetaRII was altered in rat lungs during pulmonary fibrosis induced by bleomycin. The increase in TbetaRI mRNA level was evident after 3 days of bleomycin administration, and TbetaRI mRNA continually increased for over 12 days after bleomycin instillation, whereas TbetaRII mRNA declined at day 3 post bleomycin instillation and then increased during the reparative phase of lung injury (days 8 and 12). The immunoreactivity for both TbetaRI and TbetaRII was detected in the cells of the interstitium, the epithelium, and the blood vessels of normal rat lungs. In bleomycin-induced pulmonary fibrosis, an extensive immunostaining for TbetaRI and TbetaRII was present in the cells at the sites of injury and active fibrosis. These results demonstrate that the expression of TGF-beta type I and type II receptors was altered during pulmonary fibrosis, suggesting that the TGF-beta signal transduction pathway may be involved in the pathogenesis of lung fibrosis.     

Engineering cell adhesive surfaces that direct integrin alpha5beta1 binding using a recombinant fragment of fibronectin

Integrin receptors mediate cell adhesion to extracellular matrices and trigger signals that direct cell function. While many integrins bind to the arginine-glycine-aspartic acid (RGD) motif present in numerous extracellular proteins, integrin alpha(5)beta(1) requires both the PHSRN synergy site in the 9th and the RGD site in the 10th type III repeat of fibronectin (FN). Binding of alpha(5)beta(1) to FN is critical to many cellular processes, including osteoblast and myoblast differentiation. This work focused on engineering integrin-specific bioadhesive surfaces by immobilizing a recombinant FN fragment (FNIII(7-10)) encompassing the alpha(5)beta(1) binding domains of FN. Model hybrid surfaces were engineered by immobilizing FNIII(7-10) onto passively adsorbed, non-adhesive albumin. Homo- and hetero-bifunctional crosslinkers of varying spacer-arm length targeting either the cysteine or lysine groups on FNIII(7-10) were investigated in ELISA and cell adhesion assays to optimize immobilization densities and activity. FN-mimetic surfaces presenting controlled densities of FNIII(7-10) were generated by varying the concentration of FNIII(7-10) in the coupling solution at a constant crosslinker concentration. Cells adhered to these functionalized surfaces via integrin alpha(5)beta(1) and blocking with integrin-specific antibodies completely eliminated adhesion. In addition, adherent cells spread and assembled focal adhesions containing alpha(5)beta(1), vinculin, and talin. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of biomimetic materials.     

Inhibition of transforming growth factor-beta 1 alters the growth, anchor-dependent cell aggregation and integrin mRNA expression in human promonocytes: implications for endometriosis and peritoneal adhesion formation

Transforming growth factor beta (TGF-beta) is a major secretory product of macrophages which, through autocrine/paracrine pathways, play a central role in normal reproductive tissues as well as in disorders such as endometriosis and intraperitoneal adhesion formation. Using TGF- beta antisense oligonucleotides and U937 cells (a promonocytic human cell line) as an in-vitro model, the present study examined the autocrine mediated action of TGF-beta 1 on proliferation, anchor- dependent and -independent cell aggregation and expression of several mRNAs of cell surface adhesion molecules including integrins and platelet-endothelial cell adhesion molecule (PECAM-1). Northern blot analysis and enzyme-linked inmmunosorbent assay (ELISA) revealed that treatment with TGF-beta 1 antisense, but not sense or nonsense oligomers, in a dose-dependent manner (0.1-10 microM) down-regulated the expression of TGF-beta 1 mRNA and protein to undetectable amounts at the highest antisense concentration. TGF-beta 1 antisense at < 1 mM slightly increased, while at > 3 microM significantly inhibited, the rate of DNA synthesis and proliferation of these cells (P < 0.05). Treatment with TGF-beta 1 antisense promoted cell aggregation under anchor-independent culture conditions (plastic dishes), while it suppressed colony formation under anchor-dependent culture conditions (soft agar assay). U937 cells expressed alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 1 and beta 2 integrin mRNA and PECAM-1 mRNA, while alpha v, beta 3 and beta 5 integrin mRNA was undetectable. The relative amount of alpha 2, alpha 3, alpha 4, alpha 6, beta 1 and beta 2 integrin and PECAM-1 mRNA expression were down-regulated in a dose- dependent manner after TGF-beta 1 antisense treatment, while alpha 5 integrin mRNA expression was up-regulated, although it was undetectable at 10 microM antisense. In contrast, TGF-beta 1 antisense up-regulated beta 3 mRNA expression with maximal effect occurring at 10 microM. These results provide evidence that the autocrine loop of monocyte/macrophage-derived TGF-beta 1 action is essential for regulation of growth, aggregation and the expression of adhesion molecules by these cells. We propose that in disorders such as endometriosis and peritoneal fibrous adhesions, significantly higher numbers of tissue macrophages with the capacity to express excess TGF- beta 1 yield an environment able to promote cell-cell and cell-matrix interactions, and thus lead to further complications from these abnormalities. We are currently investigating whether site-specific inhibition of TGF-beta using antisense strategy is a useful tool for management of these lesions, particularly after their post-surgical removal.      

当归注射液及前列腺素E_2对结缔组织生长因子在大鼠肺纤维化模型中表达作用的比较研究

目的探讨结缔组织生长因子(CTGF)在大鼠肺间质纤维化中的表达及其在25%当归注射液、前列腺素E2(dmPGE2)的肺脏保护作用中的意义。方法SD大鼠随机分为对照组、模型组、当归组、PGE2组。按博莱霉素(BLM)5mg/kg体重复制大鼠肺纤维化模型,当归组、dmPGE2组术后分别给于25%当归注射液(腹腔内注射)、dmPGE2(肌肉注射),于第28天处死全部大鼠。制备大鼠肺组织切片,用原位杂交及免疫组化SABC法分别检测CTGF mRNA、纤维连结蛋白(FN)和Ⅲ型胶原(Col-Ⅲ)在肺内的表达,HE染色评价肺纤维化程度。结果模型组CTGF mRNA、FN和Col-Ⅲ的表达及肺纤维化程度明显高于对照组(P<0.01),而当归组及PGE2组明显低于模型组(P<0.01)。两治疗组间比较,除CTGF外,其余指标无显著性差异(P>0.05),各组指标的相关分析表明,CTGF表达水平与肺纤维化程度(r=0.886;P<0.01)、FN(r=0.836;P<0.01)和Col-Ⅲ(r=0.918;P<0.01)呈正相关关系。结论25%当归注射液可能通过下调CTGF而减轻肺间质纤维化。